DESCRIPTION: An essential process for efficient nutrient digestion, absorption and assimilation is the constant replacement of mature intestinal epithelial cells by differentiating stem cells. The influence of nutrients as agonists that might affect this maturation process has been little explored. We have previously shown that oleic acid affects this maturation in a tissue culture system, developed in our laboratory, derived from intestinal cells (hBRIE 380i cells) which undergoes many aspects of mucosal differentiation. Dietary oleic acid also directly stimulates the secretion of a regulatory peptide, peptide Y, in the intestine at a concentration sufficient to induce differentiation and migration, in vitro, of cells in this system. PYY induces the expression of the membrane spanning protein (tetraspanin) CD63 in these cells. CD63 and other tetraspanins have been demonstrated to be important facilitators of cell adhesion and motility and to associate with proteins that interact with the ECM. The hBRIE 380i cells transfected with CD63 antisense cDNA fail to undergo differentiation. We propose that regulatory peptides mediate dietary free fatty acid induced mucosal differentiation by modulating interactions between the cell cytoskeleton and its extracellular matrix. As a model we propose to examine the mechanism by which PYY regulates stem cell maturation by answering the following questions: 1) Is PYY induction of CD63 necessary for mucosal stem cell differentiation; 2) does the effect of PYY and CD63 on cell maturation require an intact extracellular matrix; and 3) is this biological activity mediated by the Y1 and Y2 receptors? In these studies we will use hBRIE 380i CD63 antisense cells and CD63 gene disrupted mice to determine if CD63 is obligatory for mucosal cell differentiation by PYY and on signals from the ECM. We will determine plasma membrane and basement membrane proteins known to be associated with CD63 by utilizing immunoprecipitation, electron and light immunocytochemistry, and cell footprinting techniques. We will determine if PYY S action is through Y1 and Y2 receptors by using receptor ligand analogues and through antisense studies. In the present proposal we hope to elucidate a mechanism(s) by which dietary free fatty acids alter epithelial cell differentiation by modulating tetraspanin mediated cell mesenchymal interactions through the release of regulatory peptides. These studies will further expand the role of nutrients from metabolic and structural substrates to signal molecules closely linked to the feedback regulation of their own assimilation.